Isolation of the porcine ileal intrinsic factor receptor by sequential affinity chromatography

An extract containing solubilised receptor was passed through four columns containing Sepharose to which had been covalently coupled anti-cobalophilin IgG, vitamin B-12-intrinsic factor, vitamin B-12, and free intrinsic factor, respectively. Following a wash the receptor was eluted with EDTA, then residual Triton X-100 micelles and vitamin B-12-intrinsic factor were removed by Sephadex G-200 filtration. The receptor was purified 84 000-fold, sodium dodecyl sulphate electrophoresis indicated two subunits and gel filtration of its vitamin B-12-inttrinsic factor complex resolved it into two molecular species.     

Isolation and in vitro expression of the ricin A-chain gene: effect of deletions on biological activity.

The gene encoding the ricin A-chain was isolated and subcloned into an in vitro expression vector downstream from the SP6 promotor. mRNA encoding the A-chain strongly inhibited the translational activity of reticulocyte lysates. The inhibition correlated with glycosylase activity on rRNA, and could be abolished by addition of antibodies specific for ricin. mRNA generated after linearization of the vector at unique restriction sites within the A-chain coding sequence did not inhibit, except after linearization with ScaI. Also mutants lacking the 28 N-terminal amino acids of native A-chain strongly inhibited the lysates. However, in both cases no glycosylase activity could be observed. We also show that the lack of a stop codon in mRNA does not affect the level of expression as assayed here.     

2-Amino-2-deoxygalacturonic acid in lipopolysaccharides from Pseudomonas aerugimosa.

2-Amino-2-deoxygalacturonic acid was identified as a component of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 8505. The compound probably occurs in the region of polysaccharide responsible for O-antigenic specificity.     

Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration

Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 x 10(6) cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 10(7) or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected.